Vaccines and immunotherapeutics derived from the human immunodeficiency virus ( HIV) trans-activator of transcription protein for the treatment and prevention of HIV disease

ABSTRACT

Anti-lentivirus vaccines and immunotherapeutics and methods for preparing and using same are disclosed. The vaccines and immunotherapeutics are produced using non-immunosuppressive lentivirus trans-activator of transcription (Tat) proteins. An associated in vitro ultra-sensitive macrophage Tat bioassay is disclosed for assessing the immunosuppressive qualities of the lentivirus Tat preparations of the present invention. Additionally, a related long-term T4 cell propagation system for characterizing lentivirus Tat is also disclosed. The present invention has additional utility in the treatment and prevention of AIDS.

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/148,526, filed Aug. 12, 1999.

FIELD OF INVENTION

[0002] This invention relates generally to vaccines andimmunotherapeutics used to treat and prevent diseases caused bylentiviruses and methods for producing and using same. Specifically,this invention relates to vaccines and immunotherapeutics used to treatand prevent diseases caused by the Human Immunodeficiency Virus (HIV)and methods for producing and using same. More specifically, the presentinvention relates to vaccines and immunotherapeutics derived from HIV'strans-activation of transcription (Tat) proteins. In addition, relatedmethods for assessing the immunosuppressive activity of Tat aredisclosed.

BACKGROUND OF THE INVENTION

[0003] Human Immunodeficiency Virus (HIV) disease has killed over 12million people world wide since it was first recognized in 1981. Today,it is estimated that over 31 million people are infected with HIV andnearly 16,000 new infections occur daily. The Untied Nations HIV/AIDSsurveillance committee estimates that over 40 million people will beinfected by the year 2000, the majority of these new infections willoccur in developing countries. HIV disease, which includes acquiredimmune deficiency syndrome (AIDS), is caused by a virus belonging to thegroup Retroviridae (retroviruses). Specifically, HIV is a lentiviruswhich is a genus of the Retroviridae.

[0004] Many developing countries are confronted with health care issuessuch as malaria and tuberculosis (TB) which combine to kill more peopleannually than HIV. The World Health Organization can provide effectivetherapies against TB for 36 US dollars per person, and malaria can betreated for as little as $1.00 a month. However, even these seeminglyinsignificant amounts can cause severe economic hardship for individualsand families in developing countries. When the costs of these treatmentsare compared to the $12,000/individual/year cost associated with newcombination drug therapies referred to as HAART (highly activeanti-retroviral therapy), it becomes evident that HAART is an economicimpossibility in developing countries where effective anti-retroviraltherapy is needed most.

[0005] Combination drug therapy for HIV began to replace monotherapy(single drug treatments) in early 1996, and by 1999, the Food and DrugAdministration (FDA) had approved 11 drugs which could be used invarious HAART protocols. These eleven drugs are broken down into threeclasses which include nucleoside reverse-transcriptase inhibitors (NRTI)divided into two sub-groups A and B, non-nucleosidereverse-transcriptase inhibitors (NNRTI) and protease inhibitors (PI).Current recommendations for combination drug therapy include two NRTIs(one A and one B) combined with either a PI or an NNRTI. Thiscombination drug therapy has proven to be highly effective insignificantly reducing viral load (the amount of HIV present in theblood or tissues of an infected person) and preventing the onset ofsevere immune deficiency in many compliant patients (patients who taketheir medications exactly as directed). However, there are significantdrawbacks associated with combination drug therapy.

[0006] For many patients the toxic side effects diminish their qualityof life to such an extent that they simply stop taking theirmedications. For others, the therapeutic schedules are so complicatedand inconvenient that they find compliance nearly impossible to fit intoa normal lifestyle. Still, many infected persons do not benefit fromcombination drug therapy due to virus strain variation and other unknownfactors. Other patients experience excellent results initially, but dueto mutations in the virus, they suffer viral load relapses in spite offull compliance with the therapeutic regime. Many of these patients canbe treated with other drug combinations that knock the viral load backdown, but the risk of mutation, repeated drug failure, and the onset ofnew drug side effects are persistent fears. In total, the side effects,psychological burden, cost and uncertainty of efficacy continue to takea steep toll on patients currently undergoing combination drug therapy,rendering the best available option for treating this deadly virus worsethan the disease for many people.

[0007] Another significant limitation of combination drug therapy isthat these treatment regimes do not completely eliminate the virus fromthe body. Due to the complex nature of our immune system and humanretrovirus genetics, HIV is able to sequester itself inside dormantimmune cells where it remains unaffected by the drugs. Consequently, ifthe patient stops taking the medications, a rapid resurgence in HIVviral load occurs, requiring the patent to take anti-retroviral drugsfor life. Moreover, studies have demonstrated that seropositive (HIVinfected) compliant patients who have undetectable virus in their bodyare still capable of transmitting the virus sexually or through contactwith their blood. Ultimately, the best defense against any disease isprevention, and presently the best prophylaxis against the threats ofinfectious agents is vaccination.

[0008] Medical researchers have been seeking an effective HIV vaccinesince the virus was first discovered in 1983. Previous vaccine effortshave included inactivated whole virus, structurally modified,inactivated whole virus, viral sub-unit vaccines including gag (groupassociated, or core antigens), pol (viral polymerases), and env (viralenvelope antigens). In the latter group, both native and recombinantproteins have been investigated. Various vaccination techniques,including frequency of administration, routes of administration andadjuvant mixtures (inert ingredients mixed with the viral antigens tohelp stimulate the host response), have been tried. Many of thesevaccine approaches have elicited detectable immune response in animalsincluding humans, and a few have afforded the animal with limitedprotection against infection after being challenged with live virus.However, a safe and effective HIV prophylactic suitable for widespreadhuman use remains elusive. Therefore, there is a pressing need for acost effective, non-toxic, highly active treatment for HIV infectedindividuals, and even more importantly, for an effective prophylacticvaccine.

[0009] Recently, significant advances have been made in understandingthe HIV disease process. For many years, researchers have been unable toexplain the seemingly immediate and profound destruction of the immunesystem following the initial HIV infection. Equally puzzling was aphenomenon seen in a few patients referred to as long termnon-progessors (LTNP). In LTNP patients, viral loads are high and thevirus can be isolated easily from the HIV target immune cells such asCD4+ T lymphocytes (referred to herein as T4 cells). However, unlike themajority of infected individuals who develop AIDS, the LTNP do notdemonstrate significant reduction in their T4 cells and do not progressto AIDS.

[0010] One possible, non-binding, theory that may explain these twophenomena involves a non-structural protein (a protein encoded by thevirus genome that is not actually part of the virus itself) calledtrans-activator of transcription, or Tat for short. Tat is a variableRNA binding peptide of 86 to 110 amino acids in length that is encodedon two separate exons of the HIV genome. Tat is highly conserved amongall human lentiviruses and is essential for viral replication. Whenlentivirus Tat binds to the TAR (trans-activation responsive) RNAregion, transcription (conversion of viral RNA to DNA then to messengerRNA) levels increase significantly. The Tat protein associated withlentivirus virulence will be referred to hereinafter at C-Tat, or“conventional Tat.” Recently, it has been demonstrated that C-Tatincreases viral RNA transcription and it has been proposed that C-Tatmay initiate apoptosis (programmed cell death) in T4 cells andmacrophages (a key part of the body's immune surveillance system for HIVinfection) and possibly stimulates the over production of alphainterferon (αINF is a well established immunosuppressive cytokine).These, and other properties of lentivirus C-Tat proteins, have led toconsiderable scientific interest in C-Tat's role in pathogenesis and tothe present inventor's proposal that Tat may act as a powerfulimmunosuppressant in vivo.

[0011] A potential key to lentivirus C-Tat pathogenesis may involve inits ability to trigger apoptosis. Conventional Tat initiates apoptosisby stimulating the expression of Fas Ligand (FasL)(a monomericpolypeptide cell surface marker associated with apoptosis) on the T4cell and macrophage surface. When FasL is cross linked by binding withFAS (the counter part to FasL which is also expressed on a wide varietyof cell types), the apoptotic system is activated. Consequently, thedeath of these essential T4 cells and macrophages is accelerated,resulting in extreme immunosuppression. Thus, extracellular C-Tat'spresence early in the course of HIV infection could reduce a patient'simmune response, giving the virus an advantage over the host.Furthermore, the direct destruction of T4 cells and induction of αINFproduction could help explain the lack of a robust cellular immuneresponse seen in AIDS patients, as well as accounting for the initialprofound immunosuppression.

[0012] Further support for this concept is found in a surprising newobservation made by the present inventor who has demonstrated the Tatprotein isolated from long term non-progressors is different from C-Tatfound in AIDS patents. The Tat protein found in LTNP is capable oftrans-activating viral RNA, however, LTNP Tat (designated herein afteras IS-Tat for immuno-stimulatory Tat) does not induce apoptosis in T4cells or macrophages and is not immunosuppressive. Moreover, T4 cellsinfected ex vivo with HIV isolated from LTNP (such cell lines aredesignated Tat TcL) can result in the over expression of IS-Tatproteins, often to the virtual exclusion of other viral proteins, thatare strongly growth promoting rather than pro-apoptotic. The tat genescloned from these Tat TcLs reveal sequence variations in two tatregions, at the amino terminus and within the first part of the secondexon. These surprising discoveries could help explain why HIV infectedLTNP T4 cells do not die off at the staggering rate seen in HIV infectedindividuals that progress to AIDS.

[0013] The present inventor has demonstrated that macrophages areapproximately 1000 times more sensitive to Tat (both C-Tat and IS-Tat)than T4 cells which has led to the development of a new in vitroultra-sensitive macrophage Tat bioassays which permit, for the firsttime, the direct in vitro measurement of C-Tat's immunosuppressivecapacity. Using this new ultra-sensitive macrophage bioassay, thepresent inventor has been able to directly measure immunosuppressantactivity of different Tats which aided the inventor in developing a newHIV vaccine strategy.

[0014] This new vaccine strategy uses non-immunosuppressive Tat (eitherIS-Tat or denatured C-Tat that has been rendered non-immunosuppressive),either alone or in combination with other HIV proteins, and with orwithout an adjuvant, to provide protection against HIV infection. Thispreparation can also be used as an immunotherapeutic in HIV infectedindividuals to prevent the profound immunosuppression associated withAIDS by inducing the inoculated person's immune system to produceneutralizing antibodies directed against indigenous C-Tat (Tat producedas a result of natural HIV infection).

[0015] The use of C-Tat protein as a potential HIV vaccine component hasbeen previously described in the medical literature; however, thesestrategies have been either unsuccessful or impractical for human use.Previously published efforts required multiple inoculations over aprotracted time period in order to induce an immune response. This mayhave been caused by the immunosuppressive activity of the C-Tatcomponent of the vaccine itself. These previous investigators did nothave a convenient and reliable in vitro assay system in which to assesstheir C-Tat preparations for immunosuppressive activity.

[0016] The safety testing conducted in these earlier studies looked forevidence of C-Tat toxicity in animals and trans-activation activity incell culture, but not immunosuppressive activity directly. Some forms ofC-Tat toxicity are not associated with C-Tat immunosuppression.Consequently, assays that merely measure non-immunosuppressive toxicity,such as short-term small animal models, cannot be reliably used to alertan investigator that his C-Tat vaccine preparation wasimmunosuppressive. As a result, if an immunosuppressive vaccine wasunknowingly administered to a test animal, multiple vaccinations over aprotracted time period would be required to induce an immune response.

[0017] It is yet another non-binding theory of the present inventor,that based on the observations with long-term CD4+ Tat T cell lines (TatTcL), clinical observations, and experiments in animals, attenuated Tat(more specifically IS-Tat or, alternatively, C-Tat proteins that havebeen chemically or physically altered) may act as an immune stimulantactivating T4 cells inducing their proliferation. This principle mayhelp to explain the stable T4 levels seen in LTNP. Moreover, it isproposed that attenuated Tat may be useful as an adjuvant whenco-administered with other active vaccine components such as, but notlimited to, vaccines for other viruses, bacteria, rickettsia and cancercells.

SUMMARY OF THE INVENTION

[0018] For the purposes of clarification, and to avoid any possibleconfusion, the trans-activating (Tat) proteins of the present inventionwill be referred to hereinafter as either “C-Tat” when conventionalimmunosuppressive Tat protein is intended, “ox-C-Tat” for chemicallyoxidized C-Tat, “IS-Tat” when the immunostimulatory Tat protein found inlong-term non-progressors is referred to, or “Tat” when both forms ofTat protein are included.

[0019] Therefore, it is an object of the present invention to providelentivirus vaccines which are derived from non-immunosuppressivelentivirus Tat protein.

[0020] It is another object of the present invention to produce HIVvaccines derived from a non-immunosuppressive lentivirus C-Tat.

[0021] It is another object of the present invention to produce HIVvaccines derived from IS-Tat.

[0022] It is yet another object of the present invention to produce HIVvaccines from a non-immunosuppressive recombinant Tat using the nucleicacid sequence, or portions thereof, of the IS-Tat.

[0023] It is another object of the present invention to provideimmunotherapeutics made from non-immunosuppressive lentivirus Tat.

[0024] It is another object of the present invention is to provide an invitro ultra-sensitive macrophage Tat bioassay for determining theimmunosuppressive activity of a lentivirus Tat protein.

[0025] It is yet another object of the present invention to provide arelated long-term T4 cell propagation system for characterizinglentivirus Tat that is immunostimulatory rather than immunosuppressive.

[0026] It is yet another object of the present invention to provide anadjuvant component consisting of attenuated, or suitably modified, Tatprotein, or immunostimulatory peptides derived from the Tat protein,useful in the formulation of other vaccines.

[0027] The Tat vaccines and Tat immunotherapeutics of the presentinvention are made from either inactivated (chemically or physicallyaltered to render the Tat protein non-immunosuppressant) native C-Tatderived from Human Immunodeficiency Virus (HIV) infected individuals, ornative non-immunosuppressive IS-Tat isolated from HIV infectedindividuals classified as long term non-progressors (LTNP) (collectivelyreferred to herein as Tat vaccines). It is also envisioned that Tatvaccines and/or Tat immunotherapeutics of the present invention may bemade using recombinant DNA techniques using either full or partial Tatsequences. The inactivated C-Tat, native IS-Tat and recombinant versionsthereof, are tested for immunosuppressive capacity using the in vitroultra-sensitive, macrophage Tat bioassay of the present invention.

[0028] In contrast to the prior art, the Tat vaccines andimmunotherapeutics of the present invention are made using lentivirusTat that is proven to be non-immunosuppressive using the in vitroultra-sensitive, macrophage Tat bioassay of the present invention.Consequently, the Tat vaccines made in accordance with the methods ofthe present invention do not suppress the immune system of therecipient, resulting in a vaccine or immunotherapeutic that can beadministered using vaccine protocols comparable with other commercialvaccines as opposed to prior art protocols which called for multiplevaccinations over a protracted time period.

[0029] The Tat vaccines and immunotherapeutics of the present inventioninduce the production of lentivirus Tat protein, neutralizing antibodiesin the vaccine recipient, thus protecting them from HIV infection.Individuals that are already infected with a lentivirus, specificallyHIV, benefit from the administration of the Tat immunotherapeutics ofthe present invention by producing a Tat neutralizing antibody thatreduces the immunosuppressant capacity of the indigenously produced Tat.

[0030] In another embodiment of the present invention the IS-Tat made inaccordance with the teachings of the present invention, and/or thechemically modified Tat proteins of the present invention, are used toprovide adjuvants which stimulate T4 cell proliferation and heighten theimmune response to the vaccine components.

[0031] In another embodiment of the present invention, the Tat vaccinesand Tat immunotherapeutics are also compounded with known adjuvants tofurther increase the host's immune response to the Tat proteins. Thesepreviously-known adjuvants may include, but are not limited to, alum,mineral oil, mineral oil-detergent emulsions, Freund's completeadjuvant, incomplete Freund's adjuvant, liposomes, MF59 (Chiron, Inc.,Emeryville, Calif.) IFA51 (Seppic, Inc., Fairfield, N.J.), MPL (Corixa,Seattle, Wash.) and others, just to name a few. In one embodiment of thepresent invention, oil-based adjuvants may be preferred due to theirpropensity to improve host immune response to small protein antigenssuch as Tat.

[0032] The in vitro ultra-sensitive, macrophage Tat bioassay of thepresent invention permits the direct assessment of the immunosuppressantqualities of any Tat protein. The in vitro ultra-sensitive, macrophageTat bioassays of the present invention have been developed to detect aminimum of 500 pM of C-Tat. In accordance with the teachings of thepresent invention, macrophages exposed to C-Tat are stimulated toexpress Fas Ligand (FasL) on their surfaces, the expression of which isthen detected. In one embodiment of the present invention -FasLexpression is detected using anti-FasL antibodies.

[0033] Additionally, a related long-tern T4 cell propagation systemuseful for isolating and producing lentivirus IS-Tat is also disclosed.

[0034] Further objects and advantages of the Tat vaccines, Tatimmunotherapeutics and the in vitro ultra-sensitive, macrophage Tatbioassays produced in accordance with the teachings of the presentinvention, as well as a better understanding thereof, will be affordedto those skilled in the art from a consideration of the followingdetailed description of exemplary embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] A detailed description of the invention is hereby described bynon-limiting examples with specific reference being made to the drawingsin which:

[0036] FIGS. 1A-F depicts fluorescence-activated cell sorter analysis ofhuman monocytes, enriched from peripheral blood by gradient purificationwhich have been treated in culture for six days with either no stimulus(0) (A), 100 ng/ml LPS (B), or 50 nM Tat (C). Immunofluorescence of allcells in culture following staining with a rhodamine-labeled anti-CD14antibody The CD14+ cells (M1) as a percentage of all cells in culture attime of assay: 3.6% (D), 25.7% (E), 31.7% (F).

[0037] FIGS. 2A-F depicts mouse peritoneal macrophages that wereisolated either after in vivo thioglycolate stimulation (TG+), orwithout in vivo stimulation (TG−). Mouse peritoneal macrophages werecultured for 5 days either in the absence of additional stimulation (0)(A) and (D), with LPS (100 ng/ml) (B) and (E), or with Tat (100 nM) (C)and (F). Arrow indicates population of apoptotic, annexin-reactive cellsaccumulating in unstimulated cultures.

[0038]FIG. 3 depicts median fluorescence of monocytes, cultured for sixdays either with no stimulus (0), 50 ng/ml TNFA, 100 ng/ml LPS,decreasing concentrations of C-Tat, or 50 nM oxidized ox-C-Tat andstained with an anti-FasL monoclonal antibody followed by afluorescenated goat anti-mouse polyclonal antibody.

[0039]FIG. 4 depicts neutralization of C-Tat-mediated induction of FasLon human macrophages by anti-Tat polyclonal antibodies.

[0040]FIG. 5 depicts participation of FasL in C-Tat-mediated suppressionof lymphocyte proliferation. Human PBMCs from two individuals (PBMCs #1and #2) cultured for six days in medium alone, tetanus (0.3 Lf/ml)antigen (Ag) or Candida (4 μg/ml) Ag, or Ags with additionally 125 or250 nM recombinant Tat protein.

[0041]FIG. 6 depicts human polymorphonuclear neutrophils (PBMC) from oneindividual (PBMCs #3) cultured in either medium with tetanus antigen(Ag, 0.3 Lf/ml), tetanus antigen with the further addition of 50 nMC-Tat (Ag+Tat), or tetanus antigen with 50 nM C-Tat and recombinantsoluble Fas protein (25μg/ml).

[0042]FIG. 7 depicts proliferation of polymorphonuclear neutrophils(PBMC) cultured with either tetanus (PBMCs #4 and #>5) or Candida (PBMCs#5) antigen (Ag) alone as in FIG. 6, compared to cultures in which C-Tat(Ag+Tat, 125 nM), or Tat (125 nM) and the antagonistic anti-Fasantibody, ZB4 (250 μg/ml, UBI, Lake Placid, N.Y.) were also added(Ag+Tat+αFas).

[0043]FIG. 8 depicts the absence of C-Tat-directed antibody response inimmediate seroconverters (IMSc) and progressors (P). Antibodies to Tatin sera from uninfected controls (Con), immediate seroconverters (IMSc),long term non-progressors (LTNP), random HIV-positive (HP) individuals,and progressors (P), were measured by ELISA against recombinant Tat.

[0044]FIG. 9 depicts an analysis of anti-C-Tat and anti-p24 antibodyresponses by protein immunoblot. Upper: Protein lysate from a normal Tcell line (NI TcL), lysate from a PBMC-derived T cell line engineered tooverexpress the IS-Tat protein (Tat TcL) and recombinant Tat protein(Rec Tat) were resolved by polyacrylamide gel electrophoresis. Proteinmarkers (kilodaltons, kd) and expected migration of the Tat protein(filled arrow) are indicated. Expected migration of p24 (open arrow) andC-Tat (filled arrow) proteins, and relative position of protein markers(kd) are indicated.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0045] For the purposes of clarification, and to avoid any possibleconfusion, the trans-activating (Tat) proteins of the present inventionwill be referred to hereinafter as either “C-Tat” when conventionalimmunosuppressive Tat protein is intended, “ox-C-Tat” for chemicallyoxidized Tat protein, “IS-Tat” when the immunostimulatory Tat proteinfound in long-term non-progressors is referred to, or “Tat” when bothforms of Tat protein are included.

[0046] The present invention provides anti-lentivirus vaccines andimmunotherapeutics (specially anti-Human Immunodeficiency Virus (HIV)vaccines and immunotherapeutics); for preventing and treating HIVinfection, and a related in vitro ultra-sensitive, macrophagetrans-activator of transcription (Tat) bioassay for assessing Tatimmunosuppressant activity. The vaccines and immunotherapeutics of thepresent invention can be made from a variety of Tat sources. In oneembodiment of the present invention the C-Tat is derived from cellsinfected, either naturally or experimentally, with a lentivirus,preferably a human lentivirus. In another embodiment of the presentinvention IS-Tat is derived from HIV strains associated with a class ofpatients known as long term non-progressors (LTNP) The IS-Tat isisolated from both naturally and experimentally infected cells. In yetanother embodiment of the present invention both C-Tat and IS-Tat areproduced using standard recombinant DNA techniques known to thoseskilled in the art.

[0047] The IS-Tat proteins can be isolated and characterized using along-term T4 cell propagation system developed in accordance withteachings of this invention. In this embodiment, peripheral bloodmononuclear cells (PBMC) are isolated and purified from non-infectedindividuals and infected with HIV isolated from LTNP using methods knownto those skilled in the art. By way of example, and not intended as alimitation, approximately 3×10⁶ of freshly isolated uninfected PBMCs areco-cultured with and equal number of freshly isolated PBMCs from LTNPs.These freshly inoculated PBMC/HIV cultures are diluted to a very lowdensity of approximately 100 cells per well in a 96 well tissue cultureplate (approximately 100 to 200 μL per well) and maintained in RPMI 1640containing 10% fetal bovine sera (FBS) (media) at approximately 37° C.with 5% CO₂. The cultures are monitored every 3-7 days for theappearance viable cell colonies, at which time fresh media is added. Thepresence of viable, free HIV is monitored using methods which include,but are not limited to, measuring either viral core p24 antigen or theenzyme reverse transcriptase.

[0048] Under conditions of very low cell density, cell cultureconditions which normally kills primary T4 cells (the cells which hostthe HIV infection), cell lines which profoundly over-express IS-Tatprotein are initiated (designated herein as Tat TcL). The Tat TcL celllines are rapidly expanded by culture in medium supplemented withinterleukin 2 (20 U/ml), or interleukin 4 (10 ng/ml), but most potentlyby co-culture with limiting numbers (<100) of allogeneic, PHA oranti-CD3+ anti-CD28-stimulated, peripheral blood cells. This IS-Tatdiffers from C-Tat insofar as it is growth promoting rather thanapoptotic and the Tat TcL cell lines of the present invention producedIS-Tat to the virtual exclusion of other HIV viral proteins. When usedin accordance with the teachings of the present invention, the long-termT4 cell propagation system described above permits the rapidcharacterization and robust production of Tat proteins, includingIS-Tat. Moreover, the Tat TcL of the present invention may also serve asan in vivo source of IS-Tat by directly administering the Tat TcL cellsto a patient.

[0049] Recombinant Tat can be made using methods known to those ofordinary skill in the art using expression systems which include, butare not limited to, eurkaryotic and prokaryotics cells. Non-limitingexamples of eukaryotic cells suitable for the expression of Tat proteinsinclude insect cells, yeast cells, and mammalian cells. Prokaryotic cellexpression systems include bacteria. Suitable nucleic acid sequencesinclude tat genes derived from lentivirus RNA and are available throughpublic domain gene libraries such as, but not limited to, GenBank(National Center for Biotechnology Information). In another embodimentof the present invention, the tat gene is cloned from the Tat TcL cellsdiscovered and sequenced by the present inventor.

[0050] Tat TcL genes of the present invention revel sequence variationin two regions of tat, at the amino terminus and within the first partof the second exon. Conventional Tat protein contains two prolines (P)within its first seven residues, a regulatory motif that is frequentlyobserved in proteins designated for protolytic degradation via theproteosome. The second P at residue six is followed by a consensuscharged residue at position seven for all clades (closely relatedtaxonomic sub-groups of HIV viruses designated as clades A, B, C, D,etc.), the charged residue mimicking a constitutive phosphorylation thatconventionally inhibits proteosomal degradation. For clade B C-Tat, theposition seven charged residue is arginine (R). The growth promotingIS-Tats frequently have modified the R to serine (S), an event thatwould lead to rapid proteosomal degradation of IS-Tat in the absence ofS phosphorylation. The second region of extensive variation in thegrowth-promoting IS-Tat is found in the first part of the second exonbetween residues 73 and 84. Indeed, some of these IYS-Tats have mutatedto entirely lose second exons, while C-Tat proteins as short as 86residues have been reported to be pro-apoptotic. Moreover,immunostimulatory peptides may ideally be derived from regions of theIS-Tat protein that are variant in LTNP, including but not limited to,the second exon and the amino terminus of the Tat protein.

[0051] Conventional-Tat and recombinant C-Tat are chemically orphysically inactivated in accordance with the teachings of the presentinvention prior to being incorporated into the vaccines orimmunotherapeutics of the present invention. These C-Tat proteins areinactivated to reduce or eliminate their immunosuppressant activity,which is verified using the in vitro ultra-sensitive, macrophage Tatbioassay of the present invention. The chemical and physical methods ofthe present invention used to inactivate the C-Tat include, but are notlimited to, chemical oxidation and irradiation. In one embodiment of thepresent invention the C-Tat proteins are chemically oxidized using 3%hydrogen peroxide for one hour at approximately 25° C. Other methods forchemical oxidation include: 1 mM to 1M sodium periodate for one hour atapproximately 25° C.; 1 mM to 1M peroxyacids for one hour atapproximately 25° C.; 1 mM to 1M m-chloroperbenzoic acid for one hour atapproximately 25° C.; and other chemical and physical oxidativeprocesses known to those skilled in the art. Residual oxidants can beeliminated from the C-Tat preparation by adding a suitable biocompatibleoxidizable substrate to the C-Tat preparation after the oxidation iscomplete. (Oxidized C-Tat preparations are referred to hereincolleectively as ox-C-Tat). Samples of suitable biocompatible oxidativesubstrates include, but are not limited to, glycerol, carbohydrates andsimilar compounds known to those in the art. Immuno-stimulatory Tatproteins expressed by LTNP do not require inactivation prior toincorporation onto the vaccines and immunotherapeutics of the presentinvention. However, to verify that the IS-Tat proteins are naturallynon-immunosuppressant, it is desired to test them using the in vitroultra-sensitive, macrophage Tat bioassay of the present invention.

[0052] All forms of Tat proteins, C-Tat, ox-C-Tat, IS-Tat, and theircorresponding recombinant proteins, are tested for immunogenicity inmice. The ox-C-Tat proteins produced in accordance with the teachings ofthe present invention were compared with identical vaccines composed ofun-inactivated C-Tat. It was surprisingly found that the ox-C-Tatinduced a stronger immune response in mice than non-inactivated C-Tat.Table 1 depicts the results of this study. Group I received the C-Tat,group II received ox-C-Tat. Anti-C-Tat titers were remarkably enhancedat 2 weeks and 6 weeks post immunization in the animals inoculated withox-C-Tat as compared to animals inoculated with immunosuppressant C-Tat.Further studies (data not shown) demonstrated that immunosuppressioncontinued for a minimum of 12 weeks post immunization, at which pointthe animals were sacrificed. TABLE 1 Mean Anti-C-Tat Titer MeanAnti-C-Tat Animal (SD) Titer (SD) Group Immunogen Week 2 Week I C-Tat<100 922 (822) II ox-C-Tat 12,750 (670) 17,675 (4925)

[0053] One possible, non-binding, theory offered to explain thesurprising antigenic superiority of ox-C-Tat as compared to C-Tat isthat the C-Tat may suppress antigen presenting cell (APC) activity inaddition to T4 cell function. To test this non-binding theory, a seriesof HIV viral antigens preparations were mixed with either ox-C-Tat orC-Tat and formulated into vaccines in accordance with the teachings ofthe present invention. The antigen preparations included C-Tat plus HIVp24 (a group associated antigen (gag) which makes up part of the viralcore), ox-C-Tat plus HIV p24 and controls consisting of either HIV p24alone and HIV p24 antigen oxidized using the same methods used toprepare the ox-C-Tat (ox-p24).

[0054] Vaccines were prepared using 5 μg of C-Tat protein mixed with 5μg each C-Tat and recombinant p24 (Chiron Corp, San Jose, Calif.) in 100μl of complete Freund's adjuvant administered subcutaneously in theanimals' flanks. Subsequently, sera were collected every other week forantibody response (up to ten weeks), or lymph nodes were harvested at 6weeks for T cell proliferation assays. Immune response to the vaccinepreparations were determined using T-cell proliferative assays describedbelow and measurement of specific antibody responses using anenzyme-linked immunosorbant assay (ELISA). The ELISA assays wereperformed by methods known to persons of ordinary skill in the art.Briefly, viral antigens and various Tat preparations at 1 μg/ml wereapplied to plastic, 96-microwell plates in carbonate/bicarbonate coatingbuffer pH 9.6 overnight at 4° C., and blocked overnight at 4° C. inphosphate buffered saline, pH 7.4 (PBS), with 0.05% Tween-20, 2.5%bovine serum albumin (Sigma), and 5% FBS (GIBCO, Grand Island, NY)(blocking buffer). Sera, diluted 1:100, 1:1000, and 1:10,000 into assaybuffer (PBS+0.056% Tween 9:1 blocking buffer), were incubated on thecoated plates for 1 hr at 37° C. Reactions were developed withaffinity-purified, horseradish peroxidase conjugated anti-human IgG orIgM, or anti-mouse IgG (KPI, Gaithersburg, Md.) for 30 min at 37° C.,followed by tetra methyl benzidine (TMB) substrate, and stopped in 4NH₂SO₄. Anti-p24 antibodies were measured by commercial ELISA. Plateswere read (V Max Pro, Molecular Devices, Sunnyvale, Calif.) for thedifference in optical density between 450 nM (signal) and 575 nM(background).

[0055] Animals which received the p24/C-Tat (non-inactivated C-Tat)vaccine mixture had a ten-fold weaker p24 immune response that thoseanimals receiving either p24 alone, ox-p24 or p24/ox-C-Tat. Moreover,the animals which received just ox-C-Tat had a seven fold betterresponse to the C-Tat vaccine component that those animals receivingnon-inactivated C-Tat alone. These results support the non-bindingtheory of the present inventor that C-Tat vaccines which areadministered without first inactivating C-Tat's immunosuppressantqualities results in a vaccine that acts as an overall immunosuppressantreducing the recipient's immune response to Tat and any other associatedimmunogens. Table 2 depicts the results of this study. Group I receivedp24 alone, group II received C-Tat plus p24, group III received ox-C-Tatplus p24 and group IV received ox-p24 alone. Anti-p24 and anti-Tattiters were remarkably enhanced in the animals co-inoculated withox-C-Tat as compared to animals co-inoculated with immunosuppressantC-Tat. TABLE 2 Mean Anti-C-Tat Mean Anti-p24 Titer Group Immunogen Titer(SD) (SD) I p24 0 9109 (1337) II C-Tat + p24 983 (317) 1100 (384) IIIox-C-Tat + p24 9033 (657) 8013 (1410) IV ox-p24 0 7944 (1245)

[0056] In order to better understand which cells of the immune systemwere possibly effected by the immunosuppressant activity of C-Tat, Tcell and macrophage cell populations were studied. The role of T cellsin the immune response to Tat was assessed using T cells harvested fromthe animals vaccinated with the C-Tat plus HIV p24 and ox-C-Tat plus HIVp24 described immediately above. The T cells were then exposed topurified HIV p24 antigen at concentrations of 0.02, 0.12 and 2 μg/ml.The T cells derived from the ox-C-Tat/p24 animals demonstrated a typicalrecall proliferative response (T cells populations previously sensitizedto an active immunogen such as a vaccine will increase in numbersrapidly when exposed to the same immunogen post sensitization). However,when T cell populations taken from animals immunized with C-Tat/p24 wereexposed to the same p24 preparations, the recall proliferative responsewas significantly reduced. The blunting, or reduction, in recallproliferative response seen in T cell populations taken from animalsco-inoculated with C-Tat as compared the animals receiving ox-C-Tatvaccines demonstrates the immunosuppressive action of un-inactivatedC-Tat on T cells. The significant increase in antibody response toantigens co-inoculated with inactivated C-Tat and the markedly superiorT cell proliferative response in the same animals as compared to animalsvaccinated with un-inactivated C-Tat, combine to support the utility ofthe vaccines and immunotherapeutics of the present invention.

[0057] As previously stated, the in vitro ultra-sensitive Tat bioassayof the present invention is used to assess the immunosuppressantactivity of the Tat proteins used in vaccines and immunotherapeutics ofthe present invention. This assay utilizes fresh macrophage cellssubstantially purified from human peripheral blood using standarddensity gradient enrichment procedures, or other cell isolationprotocols known in the art. The substantially purified macrophages arewashed and then cultured in cell culture media using standard tissueculture techniques. In one embodiment of the present invention, thesubstantially pure macrophages are cultured in RPMI 1640 supplementedwith 10% FBS at 37° C.

[0058] The in vitro ultra-sensitive macrophage Tat bioassay of thepresent invention is performed using a positive control (FasL inducingcompound) and a negative control (no active compound is added to theculture). Suitable positive controls include, but are not limited to,lipopolysaccharide (LPS) and/or tissue necrosing factor alpha (TNF α) ata final concentration of 100 ng/mL and at 50 ng/mL respectively. Testsamples (Tat preparations) are run at final concentrations ranging from50 pM to 50 nM and include C-Tat, ox-C-Tat, IS-Tat, Tat proteins whichhas been pre-reacted with antibodies and other combinations and Tatpreparations.

[0059] The test samples and controls are individually mixed with thesubstantially pure macrophages seeded at a density of 10⁶ cells/mL inround bottom tubes (Falcon 2059) containing RPMI 1640 with 10% FBS(herein referred to collectively as assay cultures). The assay culturesare then incubated for a suitable period of time, preferably from fiveto six days, at 37° C. An enriched carbon dioxide atmosphere may or maynot be required depending on whether optional buffering systems (e.g.HEPES) have been added to the RPMI 1640 cell culture medium. In theabsence of such optional buffering systems, a 5% carbon dioxideincubation environment is preferred.

[0060] At the end of the incubation period, cells are removed from eachassay culture and the presence of any induced FasL expression isdetected. In one embodiment of the present invention, the test andcontrol cells are washed and stained using an anti-FasL antibody.Suitable antibodies include, but are not limited to, monoclonals such asNOK-1, NOK-2, G247-4 (Pharmingen, San Diego, Calif.), mab 33(Transduction Laboratories, Lexington, Ky.), C20 (Santa CruzBiotechnology, Santa Cruz, Calif.) or MIKE-2 (Alexis, San Diego,Calif.). The cells are then washed again and stained with an appropriatedetection reagent such as, but not limited to, a fluoresceinisothiocyanate (FITC) conjugate as known to those of skill in the art.If mouse monoclonals are used, an anti-mouse FITC conjugate is preferred(for suitable non-limiting examples see the Sigma Chemicals Biochemicaland Reagents for Life Sciences Research Catalogue 1999 edition, pages1367-1370, Sigma Chemicals, St. Louis, Mo.). For anti-FasL antibodiesderived from other sources, an appropriate anti-species FITC conjugateis used. It is also within the scope of the present invention to useanti-FasL antibodies pre-conjugated to an appropriate detection reagentsuch as a fluorescent dye including FITC.

[0061] After the substantially pure macrophages have been stained withan anti-FasL antibody and the detection reagent, the cells are washedand the reaction between the anti-FasL and the surface of thesubstantially pure macrophage is determined using a detection systemsuitable for the detection reagent used. For example, if a fluorescenceconjugate detection reagent is used, the macrophage-anti-FasL reactionis detected using a fluorescent activated cell sorter (FACS) system.Control staining is performed using the fluorescent detection reagentalone and subtracted from the FasL staining seen in the assay cultures.Using the data thus obtained the number of FasL positive macrophages andthe intensity of the reaction is determined. FIGS. 1 and 2 depictscattergrams using such a FACS system. The greater the percentage ofFasL positive cells in a given assay culture, the more immunosuppressantthe compound in the assay culture is. Negative controls should alwaysremain non-reactive with the anti-FasL antibody and the positive controlshould fall within predetermined ranges. The acceptable range for agiven positive control will depend upon the control materials used, thedetection reagent, and the detection system used.

[0062] Turning now to a detailed explanation of the associated figures.FIG. 1 depicts a FACS analysis of human monocytes (MΦ), enriched fromperipheral blood by centrifugal elutriation, cultured for six days witheither no stimulation (Stim: 0, FIG. 1a), 100 ng/ml of LPS (Stim: LPS,FIG. 1b), or 50 nM Tat (Stim: TAT, FIG. 1c). The large CD 14+MΦ (M1 100CD14+) are gated in panels 1 a-c. FIG. 1d-f depicts immunofluorescenceof all cells in culture after staining with a rhodamine-labeledanti-CD14 antibody (Pharmingen, San Diego, Calif.). The CD 14+ cells(M1) are depicted as a percentage of all cells in the culture at thetime of assay (FIG. 1d=3.6%, FIG. 1e=25.7% and FIG. 1f=31.7%).

[0063]FIG. 2a-f illustrates how C-Tat promotes the viability on murineMΦ that have been previously activated in vivo. Mouse M(D richsuspensions of peritoneal cells were isolated after three days. One MΦpopulation was collected from mice stimulated with thioglycolate (TG)intraperitoneal injections (TG+, FIGS. 2a-c) and another population wascollected from un-stimulated mice (TG-, FIGS. 2d-f). The murine MO werecultured for five days either in the absence of additional; stimulation(FIGS. 2a,d), with 100 ng/ml LPS (FIGS. 2b,e), or with 100 nM C-Tat(FIGS. 2c,f) and then analyzed by flow cytometry scatter plot for alarge population of cells (gate M1, % MΦ). Arrow 201 indicatespopulation of apoptotic, annexin-reactive cells accumulating inunstained cultures. FSC=forward scatter.

[0064]FIG. 3 depicts medium fluorescent (MFI) of monocytes cultured forsix days either with no stimulation (0), 50 ng/ml TNFα, 100 ng/ml LPS,descending concentrations of C-Tat (T, A, T) or 50 nM ox-C-Tat.

[0065]FIG. 4 graphically illustrates neutralization of C-Tat-mediatedinduction of FasL on human MΦ by anti-Tat polyclonal antibodies. HumanMΦ were cultured for two days either with no stimulus, with 50 nM Tatpretreated for one hour with murine polyclonal antibodies (1:100dilution) prepared to ox-C-Tat, or with pre-immune serum (1:100).

[0066] FIGS. 5-7 depict participation of FasL in Tat-mediatedsuppression of lymphocyte proliferation. FIG. 5 illustrates humanperipheral blood mononuclear cells (PBMCs) from two different individual(PBMCs #1 and PBMCs #2) cultured for six days in medium alone, tetanusantigen (0.3 Lf/ml), Candida antigen (4μg/ml) (Ag) or Ag withadditionally 125 or 250 nM recombinant C-Tat protein, were pulsed withtritiated thymidine over the last 18 hours before harvest. Results arerepresentative of two experiments with different PBMCs. FIG. 6 depictshuman PBMCs from one individual (PBMC #3) cultured for five days ineither medium (not shown), tetanus antigen (0.3 Lf/ml) (Ag), Ag withfurther addition of 50 nM C-Tat (Ag+Tat- or Ag with 50 nM C-Tat andrecombinant soluble (s) FAS protein (25 μg/ml) to block surface FasL(Ag+Tat+sFAS). Experimentally, tritiated thymidine was added over thelast 18 hours in culture and results were graphed a stimulation index(mean counts per minute (cpm)/mean cpm medium control). Results arerepresentative of three experiments.

[0067]FIG. 7 shows proliferation of PBMCs cultured for six days witheither tetanus (PBMCs #4 and PBMCs #5) or Candida (PBMCs #%) antigen(Ag) alone as in FIG. 5, compared with cultures in which C-Tat (Ag+Tat,125 nM), or C-Tat (125 nM) and the antagonistic anti-FAS antibody(αFAS), ZB4 (250 pg/ml) (Upstate Biotechnology, Lake Placid, N.Y.) werealso added (AG+Tat+αFAS). Results are representative of threeexperiments.

[0068]FIG. 8 demonstrates that antibody responses to Tat are delayedand/or difficult to maintain. Antibodies to Tat in sera from uninfectedcontrols (Con), immediate seroconverters (IMSc), long term nonprogressors (LTNP), random HIV-positive (HP) individuals, andprogressors (P), were measured by ELISA against recombinant Tat. Thedifference in anti-Tat antibody response between LTNP and either IMSc orthe P groups is highly statistically significant (P<0.0003 versus IMSc,P<0.0024 versus P, unpaired t test), whereas the difference in antibodybetween the LNTP and the HP groups is also statistically significant(P<0.02, unpaired t test).

[0069]FIG. 9 illustrates protein immunoblots that confirm that the fiveIMScs made no anti-Tat antibodies, whereas they already made antibodiesto other viral proteins, including p²⁴ (RR open Arrow). All the LNTPsera (examples DH and SF) and a rabbit antiserum to recombinant Tat(RAB) recognized both recombinant (Rec) and native C-Tat (Tat TcL).

[0070] The inventor of the present invention has developed vaccines,immunotherapeutics and related bioassays for the treatment andprevention of lenitvirus diseases including Acquired Immune DeficiencySyndrome (AIDS). Moreover, the present invention provides methods fortreating lentivirus diseases by administering the immunostimulatoryforms of Tat proteins of the present invention. These immunostimulatoryTat proteins include, but are not limited to, recombinant IS-Tat,natural and synthetic LTNP peptide, native IS-Tat and attenuated nativeTat. Furthermore, the Tat TcL cell lines of the present invention mayalso be used as an in vivo source of immunostimulatory Tat when the TatTcL cell line is directly administer to the mammal. In addition,co-infection of a lentivirus infected mammal using a viable virusisolated from a LTNP can also serve as an in vivo source ofimmunostimulatory IS-Tat.

[0071] While this invention has been described with respect to variousexamples and embodiments, it is to be understood that the invention isnot limited thereto and that it can be variously practiced within thescope of the following claims.

I claim:
 1. A lentivirus vaccine comprising: a non-immunosuppressantlentivirus trans-activator (Tat) protein.
 2. The lentivirus vaccine ofclaim 1 wherein said lentivirus is a human lentivirus, or portionsthereof.
 3. The lentivirus vaccine of claim 1 wherein said lentivirus isHuman Immunodeficiency Virus (HIV) or portions thereof.
 4. A lentivirusimmunotherapeutic comprising: an non-immunosuppressant lentivirustrans-activator (Tat) protein.
 5. The lentivirus immunotherapeutic ofclaim 4 wherein said lentivirus is a human lentivirus or portionsthereof.
 6. The lentivirus immunotherapeutic of claim 4 wherein saidhuman lentivirus is Human Immunodeficiency Virus (HIV) or portionsthereof.
 7. The lentivirus immunotherapeutic of claim 4 wherein saidnon-immunosuppressant lentivirus Tat protein is Tat protein treated withan oxidizing agent.
 8. The lentivirus immunotherapeutic of claim 4wherein said non-immunosuppressant lentivirus Tat protein is a Tatprotein associated with HIV long term non-progressors (LTNP).
 9. Avaccine adjuvant comprising non-immunosuppressant lentivirustrans-activator (Tat) protein.
 10. The vaccine adjuvant of claim 9wherein said non-immunosuppressant lentivirus trans-activator (Tat)protein is Tat protein treated with an oxidizing agent.
 11. The vaccineadjuvant of claim 9 wherein said non-immunosuppressant lentivirustrans-activator (Tat) protein is a Tat protein associated with HIV longterm non-progressors (LTNP).
 12. A treatment for lentivirus diseasescomprising administering a non-immunosuppressant lentivirustrans-activator (Tat) protein to a lentivirus infected mammal.
 13. Thetreatment for lentivirus diseases of claim 15 wherein said lentivirusdisease is HIV disease.
 14. The treatment for lentivirus diseases ofclaim 12 wherein said HIV disease is acquired immune deficiencysyndrome.
 15. The treatment for lentivirus diseases of claim 12 whereinsaid non-immunosuppressant lentivirus trans-activator (Tat) protein isTat derived from LTNP.
 16. The treatment for lentivirus diseases ofclaim 12 further comprising administering to an HIV infected mammalviable HIV isolated from a LTNP that reproduces in said HIV infectedmammal and produces non-immunosuppressant Tat.
 17. The treatment forlentivirus diseases of claim 12 further comprising administering a TatTcL cell line which produces a non-immunosuppressant lentivirustrans-activator (Tat) protein to a lentivirus infected mammal.
 18. An invitro ultra-sensitive macrophage Tat bioassay comprising: asubstantially pure population of macrophage cells exposed to Tat; andmeasuring the said macrophage cell's expression of FasL.
 19. A long-termT4 cell propagation system comprising: a substantially dilutedco-culture of non infected peripheral blood mononuclear cells (PBMC) andPBMCs isolated from HIV infected individuals which express IS-Tat.
 20. Amethod for characterizing lentivirus Tat comprising the steps, of; a)providing a co-culture of non-infected peripheral blood mononuclearcells (PBMC) and PBMCs isolated from HIV infected individuals; b)diluting said co-culture to a very low density such that cultureconditions would normally kill primary T4 cells; c) monitoring saidco-culture for presence of Lentivirus infection; d) detecting thepresence Tat proteins; and e) determining the immunosuppressivequalities of said detected Tat protein.